Expression of the validated proteins throughout Th-cell subsets. CD4⁺ T cells isolated from umbilical cord blood were activated with plate-bound anti-CD3 and soluble anti-CD28. The only activated cells cultured with neutralizing anti-IL4 and anti-IFNγ were used as Th0 control. Cells were stimulated with IL12 (2.5 ng/mL) for initiation of Th1, IL4 (10 ng/mL) for Th2, TGFβ (10 ng/mL) for iTreg, and IL1β (10 ng/mL) + IL6 (20 ng/mL) + TGFβ (10 ng/mL) + anti-IL4 (1 μg/mL) + anti-IFNγ (1 μg/mL) for Th17 differentiation. Cells were harvested after 72 hours of culture. (A) Polarization toward different Th-cell subtypes was examined by analyzing the expression of key transcription factors of each subtype; TBX21 for Th1, GATA3 for Th2, and FOXP3 for iTreg cells with Western blotting. (B) Cytokine secretion was used to validate the selective expression of IL17A in the cells polarized toward the Th17 phenotype. The representative result from 2 experiments is shown. (C) The expression of CTSL1, ATP1B1, KDSR, and VDR was analyzed with Western blotting. (D) CD52, IL2RB, and CXCR5 were detected with flow cytometry. GAPDH detection was used as a loading control in Western blotting analysis.